This large prospective cohort study is based on individual-level data from a population exposed to a wide range of inorganic arsenic from drinking water in Araihazar, Bangladesh. Since 2000, the study has recruited more than 35,000 men and women.

Related NIEHS-Funded Projects

Projects listed supported initial development of the epidemiological resource, added study participants, collected additional information or samples from study participants, and/or used samples or datasets derived from the resource.

Identifying gene-by-environment (GxE) interactions is a central challenge in the quest to understand susceptibility to complex, multi-factorial diseases. Developing an understanding of how genetic variation alters the effects of environmental exposures (and vice versa) will enhance our knowledge of disease mechanisms and improve our ability to predict disease and target interventions to high-risk sub-populations. Unfortunately limited progress has been made identifying GxE interactions in the epidemiological setting. Most genome-wide interaction (GWI) studies rely on statistical evidence of interaction alone and are often likely to be underpowered to detect modest interactions. In this proposal, we describe a novel two-step "GxE-omic" approach that addresses the limitations of standard GWI approaches. We will apply our approach using existing genetic and molecular data from a large Bangladeshi cohort study specifically designed to assess the effect of arsenic exposure on health. We propose to search for gene-arsenic interactions by first conducting a genome-wide search for SNPs that modify the effect of arsenic on molecular ("omic") phenotypes (i.e., gene expression and DNA methylation phenotypes, measured genome-wide) (Aim 1). Using this set of SNPs that interact with arsenic to influence molecular phenotypes, we will then test SNP-arsenic interactions in relation to arsenic-related health conditions: skin lesion status and diabetes-related phenotypes (Aim 2). As a secondary aim, we will attempt to identify SNPs that interact with arsenic to influence disease but were not selected in the Aim 1 "GxE-omic" screen by conducting conventional GWI analyses of our selected clinical phenotypes, using established "two-step" statistical approaches that leverage information on gene-environment correlation in cases and controls as well as marginal gene-disease associations. By using high-quality measures of arsenic exposure and restricting analyses to SNPs with enhanced probability of interaction with arsenic, we are highly likely to overcome the limitations of standard GWI approaches. Our team is ideally positioned to accomplish these aims, as we have conducted extensive research on the health effects of arsenic exposure and genetic susceptibility to arsenic toxicity and have extensive experience in environmental epidemiology, statistical genetics, and molecular genomics. We believe there is great promise in shifting the focus of GxE research from agnostic genome-wide interaction testing to understanding how genetic variants influence humans' response to an exposure at the molecular level. Our approach has very high potential to boost power for GWI research, enabling the identification of interactions that will enhance our understanding of disease etiology and our ability to develop interventions targeted at susceptible sub-populations. Moreover, the approach described here could potentially be used to investigate GxE interactions for a wide array of exposures and disease outcomes within our ongoing longitudinal study.

Arsenic (As) from drinking water is a major public health problem affecting over 200 million people around the world. Epidemiologic research, including our own, has demonstrated associations of As with non- malignant respiratory outcomes including impaired lung function and also with pulmonary infections. While the physiological mechanisms by which As induces non-malignant lung diseases remain largely unknown, emerging evidence from animal studies suggests disruption of the immune system as a plausible mode of action. The role of As on immune mechanisms underlying respiratory dysfunction has not been evaluated in humans. In this study, we propose to examine effects of long term As exposure from drinking water on respiratory outcomes and related immune function by measuring pulmonary function tests and activated T-cell function in 630 well-characterized adults in the HEALS cohort. Additionally, we will explore a possible protective role of vitamin D, an immune modulator, on lung function in the same population. By design, we carefully selected the following three groups of individuals (210 in each) from our population cohort based on their water and urinary As levels measured at baseline in 2000 and at three follow- up visits over 3-10 years. Group 1 includes subjects consistently exposed to low water As (< 10µg/L) since baseline; Group 2 includes subjects exposed to high levels of water As (>50µg/L) at baseline who then switched to low water As; and Group 3 includes subjects consistently exposed to high water As. We will compare the study outcomes (lung function and T-cell function) across groups 1, 2 and 3 and evaluate trends across these groups. We will consider water As, urinary As and urinary As metabolites as markers of As exposure. We will also construct a Cumulative Arsenic Index (CAI) as a measure of As exposure. Findings from this highly feasible and cost-efficient study will be able to provide important data, for the first time, from a prospective population-based study on pulmonary and immune effects of low-to-moderate doses of As exposure.

Chronic exposure to inorganic arsenic (iAs) from drinking water has been associated with increased risks of preclinical and clinical endpoints of cardiovascular disease (CVD). Methylation of InAs, first generates monomethylarsonic acid (MMAV), which is metabolized to MMAIII, and secondly dimethylarsinic acid (DMAV). A growing number of mechanistic studies have shown that MMAIII is more toxic than iAs or any of the pentavalent metabolites. Incomplete methylation, indicated by a high urinary MMA%, due to high MMAIII%, has been consistently related to the risk of skin and internal cancers and more recently incidence of cardiovascular disease (CVD) and carotid intima-media thickness (cIMT), a surrogate marker of atherosclerosis. Emerging experimental data indicate that gut microbes could convert AsV to AsIII and form MMAV and MMAIII. However, no epidemiologic studies have been conducted to identify the specific gut bacterial taxa that may influence As methylation capacity and for the role of gut bacterial taxa in As toxicology. The principal investigators have established the Health Effects of Arsenic Longitudinal Study (HEALS), a well characterized cohort in Bangladesh with 30,040 participants recruited since year 2000, with extensive experience of the collection, processing, storage, and transport of biological samples, including serum, whole blood, and spot urine samples collected from > 95% study participants. In the proposed study, they will collect fecal samples from a random 600 participants of HEALS to assess the association between gut microbiome, urinary As metabolites profile, and cIMT. Gut microbiome composition in fecal samples will be characterized as relative abundance of bacterial taxa using 16S rRNA pyrosequencing. The 600 participants will be randomly selected from an ongoing study of 3K subjects for whom measurements of cIMT and urinary As metabolites are being conducted as part of the parent study (2P42ES010349-11). The proposed study is highly relevant to the goal of RFA and will be the first epidemiologic study to address 1) whether gut microbiome may have a significant impact on the ultimate As methylation capacity, and 2) whether differences in gut microbiome composition could contribute to population variation in susceptibility to cardiovascular effects of As exposure. Microbiome-focused studies are leading new research efforts with innovative approaches to identify emerging bacterial risk factors of human disease. Since bacterial profiles are modifiable, the study could generate knowledge that may lead to interventions.

Arsenic is associated with a number of health outcomes including cancers, cardiovascular, respiratory, liver, and kidney diseases, neurodevelopment, reproduction, and diabetes. Much of the epidemiologic literature supporting these associations has evaluated arsenic exposure in adulthood. While there is a growing epidemiologic literature to suggest that arsenic exposure during in utero and early childhood periods may profoundly influence disease risk in childhood, adolescence, and later life, arsenic-related clinical outcomes in childhood as well as underlying molecular pathways have not yet been fully elucidated. Arsenic has been reported to be a potent endocrine disruptor. Alterations in hormonal balance and gene deregulation in sensitive periods such as in in utero and early life may lead to clinical dysfunction or pathologies manifest in childhood and later life. In this application, we propose to conduct the first comprehensive evaluation of molecular and clinical impacts, as related to endocrine function, in children with well-characterized in utero and early life arsenic exposure. Using an already enumerated cohort of 2-7 year old children from mothers in established population-based studies in Bangladesh, we propose to conduct follow-up visits of 500 mother-child pairs to evaluate endocrine- related characteristics in the children. The proposed research will investigate the following Specific Aims: (1) to evaluate whether in utero arsenic exposure and early childhood arsenic exposure are associated with thyroid and steroid hormones in children; (2) to evaluate whether in utero arsenic exposure and early childhood arsenic exposure are associated with gene expression profiles in children; and, (3) to longitudinally evaluate whether in utero arsenic exposure and early childhood arsenic exposure are associated with endocrine-related phenotypes (i.e., linear growth, blood pressure, and insulin resistance) in children. In exploratory analyses, we will evaluate whether these associations are modified by AS3MT genotype and child sex. Our proposed study takes advantage of a unique study population and existing data to examine endocrine characteristics and gene expression deregulation in children that may be related to in utero and early childhood arsenic exposure. We expect that the results from this proposed research would make major scientific and public health contributions toward our understanding of the health effects of arsenic exposure in several ways: (1) provide novel evidence with respect to arsenic exposure in relation to endocrine function; (2) support whether endocrine dysfunction and/or gene deregulation mediate associations between arsenic exposure and endocrine-related phenotypes; and, (3) shift the existing prevention paradigm for arsenic exposed populations, which currently focuses on exposure remediation and risk reduction in adult populations towards public health interventions targeted for pregnant-women, women of child-bearing age, and children.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide and evidence has suggested that exposure to environmental toxicants can increase CVD risk. Recently, high levels of arsenic (As), a leading environmental contaminant of concern have been associated with increases in CVD incidence, mortality, blood pressure, and systemic inflammation. Arsenic's effects at lower exposure levels are uncertain and studies are limited, but exposure at these low levels may be more widespread than previously thought, as data suggest that both water and dietary sources may contribute to overall As exposure in adults and children. In this study, the candidate proposes to take a life course approach to examining the effects of a wider range of As exposure levels on CVD risk in both adults and children and identify preclinical indicators and biomarkers of susceptibility for CVD. In the K99 mentored phase, using existing data from two adult populations ranging in levels of As exposure, the candidate will explore the relationship between As exposure and biomarkers of CVD risk, while gaining expertise in molecular epidemiology. The candidate will test archived blood and urine samples from a U.S. population to examine the relationship between low level As exposure and markers of systemic inflammation, oxidative stress and endothelial function, which have been related to high levels of As exposure among adults. In a more highly exposed Bangladeshi population, the candidate will analyze a set of potential genetic susceptibility loci in relation to As exposure and their impact on blood pressure measurements over time. She will also advance her knowledge of molecular and cardiovascular disease epidemiology through formal coursework, workshops and guidance from a diverse advisory committee of respected researchers. In the R00 independent phase, she will apply her findings to initiate a new line of investigation testing whether in utero and/or early life As exposure influences early indicators of CVD risk in two populations of children, as pregnancy and early childhood are particularly vulnerable periods when environmental insults may impact development and thus later risk of disease. In conjunction with the Health Effects of Arsenic Longitudinal Study in Bangladesh and the New Hampshire Birth Cohort Study, the candidate will implement two parallel investigations of early indicators of CVD risk among five-year-old children exposed to As. She will test whether non-invasive measures of endothelial function are associated with varying levels of in utero and/or early childhood As exposure in these two populations, as subclinical effects of As on CVD risk may occur far in advance of clinical presentation. Developing insight into the mechanisms underlying As-related CVD and identifying biomarkers of CVD risk across life stages as they relate to As exposure, will help to define the contribution of environmental As exposure to different phases of disease, from initiation to clinical presentation. With this proposed study, the candidate is positioned to take advantage of existing resources to in order to develop independent, yet complementary projects, designed to help to fill critical gaps in our understanding of the lifelong cardiovascular health impacts of a range of As exposures levels.

The overall purpose of this NIH Pathway to Independence Award is for Dr. Hall to gain the additional training needed to develop into an independent investigator capable of conducting studies to investigate interactions between nutritional and environmental factors. Building upon a background in nutrition and epidemiology, the mentored phase of this award will provide training in 1) study management and oversight, 2) advanced nutritional biochemistry and the mathematical modeling of one-carbon metabolism, and 3) laboratory sciences. The acquired skills will be applied to the current area of research, investigating the influence of nutrients involved in one-carbon metabolism on the methylation of both arsenic (As) and genomic DNA. Specifically, the roles of two nutrients, choline and betaine - i.e. key nutrients involved in methylation pathways - which have not previously been considered in relation to As and DNA methylation, will be examined. Roughly 140 million people in over 70 countries are chronically exposed to As- contaminated drinking water at concentrations far exceeding the World Health Organization standard of 10 µg/L. As is a class I carcinogen known to cause cancers of the skin, bladder, and lung, as well as ischemic heart disease and neurologic impairments. Methylation of ingested inorganic arsenic (InAs) to methylarsonic- (MMA) and dimethylarsinic acids (DMA) relies on nutrient-dependent one carbon metabolism and facilitates urinary As elimination. Methylation of DNA via one-carbon metabolism is an epigenetic modification that plays critical roles in the regulation of gene expression and maintenance of chromosomal stability, and may play a role in the underlying mechanism of As-induced carcinogenesis. In the mentored phase, the influence of choline and betaine on methylation of As and DNA will be investigated in an existing cross-sectional study of 375 Bangladeshi adults exposed to a wide range of As concentrations in drinking water. In the independent phase, the skills acquired during the training will be applied to further investigate the roles of choline and betaine, including their interactions with other nutrients, i.e. folate and creatine, in the methylation of both As and DNA utilizing data and biospecimens from a randomized controlled trial of 600 individuals chronically exposed to As. In addition, mathematical models of one-carbon metabolism will be employed to conduct in silico experiments in conjunction with the clinical trial. These experiments will 1) allow the models to be further validated against the clinical trial data and 2) provide a deeper mechanistic understanding of the results from the trial. At the conclusion of this award, Dr. Hall will have developed into an independent multidisciplinary investigator well positioned to develop additional hypotheses related to the role of nutrient/environment interactions in disease development.

Chronic arsenic exposure from drinking water is a serious public health issue in the U.S. and worldwide, as nearly 200 million people worldwide are chronically exposed to this class I human carcinogen. Arsenic increases risk for a wide array of human diseases, including cancers of the skin, bladder, and kidney; however, the mechanisms of arsenic toxicity are unclear. We hypothesize that telomeres may play a critical role in cancer-related arsenic toxicity. Telomeres are central to the regulation of genome stability, and arsenic has detrimental effects on telomeres and telomerase, the primary telomere maintenance enzyme, in a wide variety of experimental settings. In this study, we propose to characterize the relationships among arsenic exposure, various telomere-related traits, and arsenic-related outcomes in a highly feasible and efficient manner, using existing data from a longitudinal cohort study of As exposure in Bangladesh (n=27,000) with ~6-8 years of follow-up data. The proposed study uses a case-cohort design, in which we sample individuals from the cohort study to create three case groups (1,000 incident skin lesion cases, 200 incident non-melanoma skin cancer (MNSC), and 400 deaths) and a single control group (a random sample of 1000 cohort members). Using prospective blood samples, we will measure telomere length, serum markers of telomere dysfunction, and telomerase expression (for a subset with available RNA). Using baseline arsenic exposure data measured in drinking water, urine, and blood, we will assess the effect of arsenic on the measured telomere-related traits among controls and each case group. We will also assess the association between baseline telomere-related traits and our three outcomes of interest. In a second component of this work, we will measure telomere length and telomerase expression in MNSC tissue and adjacent normal tissue obtained from skin biopsies carried out for MNSC cases in this cohort. Telomere-related traits will be compared between normal and cancerous skin and among cancer subtypes, and the association between arsenic exposure and telomere- related traits in skin will be assessed. Finally, we will use existing genome-wide data on germline genetic variants to identify genetic determinants of our telomere-related traits. Several secondary aims will also be addressed. We will (1) determine if telomere-related traits mediate the effect of arsenic on health outcomes, (2) determine if arsenic effects telomere attrition rate, and (3) test telomere-related traits for association with blood levels of antioxidants. This project is a critical step towards understanding the mechanisms of arsenic toxicity in humans. The utility of all telomere-related measures derived from this work will increase as additional follow-up data accumulates in this ongoing cohort study, allowing further study of cancer, cardiovascular disease, and other arsenic-related chronic diseases.

The objective of the proposed research is to develop theory and methods for testing for sufficient cause inter- actions. The methods will be useful in identifying mechanistic interactions in biological systems and in and in the analysis and interpretation of studies in genetic epidemiology of gene-gene and gene-environment interactions. It is well known both that the presence of an interaction in a statistical model depends on the model being employed and furthermore that a statistical interaction need not correspond to an interaction in any biologically or physically meaningful sense. The sufficient cause framework makes reference to the actual causal mechanisms, referred to as sufficient causes, involved in bringing about the outcome. When two or more binary causes participate in the same causal mechanism, synergism is said to be present. Sometimes synergism cannot be identified from data; when data do imply that synergism must be present then a sufficient cause interaction is said to be present. The theory and methods developed through the proposed research lead to empirical tests for sufficient cause interactions and thus constitute tests for the joint presence of two or more causes in a single causal mechanism. The aims of the research are to extend the theory concerning the sufficient cause framework for dichotomous exposures, to develop theory for sufficient cause interaction for ordinal and categorical exposures, to develop multiply robust semiparametric tests for the presence of sufficient cause interactions, and to characterize those forms of exposure misclassification for which tests for sufficient cause interactions yield valid conclusions. The research will provide a set of techniques that can be used to identify mechanistic interactions in biological systems and will develop both a theoretical framework in which to conceptualize these mechanistic interactions and provide methods to empirically test for such interactions. The research will be useful in identifying mechanistic gene-gene and gene-environment interactions which could increase our understanding of genetic mechanisms. The implications of the research on sufficient cause interactions for understanding the mechanistic implications of standard gene-gene and gene-environment interaction tests and study designs will be explored and the methods developed will be applied to several data sets in the Health Effects of Arsenic Longitudinal Study. The research will make important advances to the statistical literature on the concept of interaction and on the implications of measurement error for causal inference. The overall research program will contribute to our understanding of the concepts of causation which form the foundation of the statistical literature in causal inference and which are being employed in medicine, epidemiology, psychology, genetics, computer science, philosophy, sociology, education and economics.

Current estimates indicate that as many as 100 million people in over 70 countries are drinking water with arsenic (As) concentrations up to 100 times the World Health Organization (WHO) guideline of 10 ug per liter. There is significant variability in progression from As exposure to clinical manifestations of disease, and it is thought that genetic and nutritional factors may together account for a substantial portion of this variability. Thus, this proposal seeks to develop biomarkers that predict risk for the development of arsenicosis. Ingested inorganic As (InAs) is methylated to methylarsonic (MMA) and dimethylarsinic (DMA) acids via folate- dependent one-carbon metabolism. A reduced capacity to fully methylate As to DMA is associated with reduced urinary elimination of As. DMAV is less toxic and has a much shorter circulating half-live than InAs, and it is rapidly excreted in urine. There is great inter-individual variability in As methylation capacity, and epidemiological evidence, including our own, suggests that people who are "good methylators" are at reduced risk for As-induced skin lesions, cancers and cardiovascular disease. However, a growing body of evidence from in vitro and laboratory animal studies indicates that the trivalent form of MMA, a requisite intermediate in the pathway toward DMA synthesis, is extremely toxic. Our work over the past several years in Bangladesh has made seminal findings regarding the strong impact of nutritional regulation of one-carbon metabolism on the inter-individual variability in As methylation, blood As levels, and risk for arsenic-induced skin lesions, e.g., our clinical trial demonstrated that folic acid supplementation lowered blood As and blood MMA concentrations. Our 1st aim is to conduct a nested case-control study of 1,000 incident premalignant skin lesion cases and 1,000 controls to test the hypotheses that the capacity to methylate As is influenced by As methyltransferase (AS3MT) genotypes and that AS3MT genotypes and As methylation are associated with risk for skin lesions. Our 2nd aim will examine whether polymorphisms in one-carbon metabolism candidate genes are associated with hyperhomocysteinemia (which is very common in this population, and is associated with reduced capacity to methylate As to DMA), with methylation of As, methylation of leukocyte DNA, and with risk for skin lesions. We will also examine whether these associations are influenced by folate status. Collectively, these aims will allow us to identify of a set of biomarkers (As metabolites, leukocyte DNA methylation, folate, B12, Hcys, and SNPs) to identify sub-groups of individuals who may be at increased risk for As-induced pathology utilizing biological specimens that are already in hand. These studies will also resolve the critical issue of whether or not a higher capacity to methylate As is beneficial. Results of these studies will create opportunities for evaluation of interventions among high risk groups. They will also result in the identification of biomarkers to facilitate targeted therapies that exploit the mechanisms involved in susceptibility, resulting in low-risk, low-cost therapeutic strategies that could potentially reduce disease risk for hundreds of thousands of people.

Cardiovascular disease (CVD) is the leading cause of death worldwide. Recent experimental studies support the hypothesis that As exposure leads to oxidative stress and vascular inflammation, a central mechanism to the development of atherosclerosis and CVD. Studies of other health effects of As exposure have suggested effect-modification by As methylation capacity and genetic susceptibility. However, epidemiologic studies of genetic susceptibility to the effects of As exposure on CVD risk are lacking. In the year 2000, we established the Health Effects of Arsenic Longitudinal Study (HEALS), a prospective cohort study of 11,746 participants (original cohort), in Araihazar, Bangladesh. In 2007, HEALS recruited another 8,288 participants (expansion cohort) to include a total of 20,034 participants. More than 90% of the cohort have exposed to As exposure at low-to-moderate levels (<300 5g/L), providing us with a unique opportunity to assess health effects of As exposure from drinking water at the levels of public health interest. As part of the parent study, cardiovascular outcomes of the cohort participants are being ascertained, and carotid artery intima-medial thickness (IMT) is being measured for 1,160 participants randomly selected from the original cohort. On the basis of these resources, our substantial pilot data, as well as cohort analyses which show a positive association between As exposure and CVD incidence and mortality, we propose a series of analyses to assess the genetic susceptibility to the effects of As exposure on the risk of atherosclerosis and CVD. We will evaluate whether the cardiovascular effects of As exposure differ by polymorphisms in genes related to As methylation (GSTM1, GSTT1, GSTO1, GSTP1, MTHFR, and AS3MT genes) and genes related to oxidative stress (NOS3, SOD2, and CYBA) and inflammation/endothelial dysfunction (TNF, IL6, ICAM1, and VCAM1) using a cross-sectional study of IMT with the subcohort of 1,160 participants, and a case-cohort study of CVD risk with 692 cases and the same subcohort of 1,160 participants from the original cohort. The strongest gene- As interaction will be tested again in a second case-cohort study with 305 cases and another subcohort of 520 participants selected from the expansion cohort. To further characterize the underlying mechanisms by which As exposure causes CVD, we will conduct a cross-sectional study with 300 subjects to evaluate the associations between As exposure and serum/urinary phenotypic markers for oxidative stress and inflammation. The proposed study will provide valuable knowledge about the pathophysiology and mechanism by which As exposure may lead to CVD and may also lead to improved prevention and risk assessment of As exposure.